Enzymes in biological systems perform important functions for the biological systems and, therefore, are important targets for pharmacological intervention. Many enzyme inhibitors have been developed and used to intervene with the biological processes catalyzed by the enzymes. An enzyme inhibitor is a molecule, which binds to enzymes and decreases their activity. The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible, causing reversible or irreversible inhibition. Reversible inhibition can be competitive, non-competitive or a combination of the two. Irreversible inhibitors bind to the enzyme normally through an irreversible covalent bond.
Many enzyme inhibitors have been developed and successfully used as pharmaceutical drugs for treatment of various diseases. Imatinib mesylate, which is the active ingredient of the brand name drug GLEEVEC™, inhibits a protein-tyrosine kinase, over-expression of which causes chronic myeloid leukemia. The enzyme inhibitor inhibits the activity of the protein-tyrosine kinase and thus effectively treats the leukemia. Specific protease inhibitors have also been successfully used to treat infections of human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Use of antibiotics to treat bacterial infections has saved countless lives. The mainstay of pharmaceutical antibiotics is beta lactams, which include penicillins, cephalosporins, monobactams, and carbapenems. However, many bacterial species develop beta lactamases, which degrade these beta lactam antibiotics and thus confer bacterial resistance to these antibiotics. Several beta lactamase inhibitors, including clavulanate, sulbactam, and tazobactam, have been developed to overcome bacterial resistance to antibiotics.
Likewise, current mainstay of therapeutic intervention for influenza is a new class of pharmaceuticals known as neuraminidase inhibitors, which inhibit the activity of influenza viral neuraminidase. Since influenza viral neuraminidase is an essential enzyme for both Type A and Type B influenza viruses, these drugs are efficacious in treating influenza. Two such drugs—oseltamivir and zanamivir—have now been approved by many countries for treating patients with influenza. Oseltamivir and zanamivir are the active ingredients of anti-influenza drug TAMIFLU and RELENZA, respectively.
Therapeutic use of these enzyme inhibitors can lead to genetic alteration of the target enzymes such that the inhibitors could no longer effectively inhibit the enzymes, a phenomenon commonly known as drug resistance. For example, use of HIV protease inhibitors can quickly lead to selection of mutations in the protease gene, which confer resistance of HIV to these inhibitors. Similarly, influenza virus mutants that confer resistance to either oseltamivir, an important drug for treatment of influenza, have appeared. During the 2008/2009 seasonal influenza, nearly all of the H1N1 isolates circulating in U.S. were resistant to oseltamivir.
It is thus important to have rapid and easy-to-use tests for detection of resistance to these enzyme inhibitors. Resistance to enzyme inhibitors is normally measured by IC50, the inhibitor concentration at which 50% of the enzyme activity is inhibited. Measurement of an IC50 value of an inhibitor for an enzyme normally involves the use of an enzyme inhibition assay comprising 8 to 12 reactions, which contain increasing concentrations of the inhibitor to be tested. The IC50 value is indicative of whether the enzyme is resistant, or the degree of susceptibility, to the inhibitor, particularly when compared to that of a known wild-type or mutant enzyme. For example, the oseltamivir carboxylate IC50 value of a wild-type H1N1 influenza virus is normally less than 5 nM whereas that of a mutant carrying an H274Y mutation in the neuraminidase gene is normally greater than 50 nM.
Because an enzyme inhibition assay normally uses 8-12 reactions, it is cumbersome and expensive to measure the IC50. Consequently, the enzyme inhibition assay has not been widely used for clinical purposes. In an embodiment of the present invention, only two reactions, one with the drug and another without the drug, are sufficient in identifying drug resistance by simply using the signal ratio of the two reactions as the indicator. The two reaction assay greatly simplifies the drug resistance detection assays. Use of a detection device with dual sample reading capability further simplifies the assay procedure.